ARID1A mutations in endometriosis-associated ovarian carcinomas

Abstract

Background: Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described.

Methods: We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas.

Results: ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions.

Conclusions: These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

Figure 1. Mutations Found in ARID1A and the BAF250a Protein It Encodes

The 20 exons of ARID1A are represented (as numbered gray boxes) above a schematic of the BAF250a protein (the blue segment, with the ARID [AT-rich interactive domain] DNA-binding domain in pink, the HIC1 [hypermethylated in cancer 1] binding domain in green, and the three C-terminal leucine-rich LXXLL motifs that facilitate interaction with gluco-corticoid receptor in yellow). The nucleotide mutations (with corresponding amino acid mutations in parentheses) listed above the schematic are those identified by means of transcriptome sequencing (RNA sequencing) of the 18 samples of ovarian clear-cell carcinoma and the TOV21G cell line in the discovery cohort, and those listed below the schematic were identified in subsequent validation efforts with the use of targeted exon resequencing and Sanger sequencing of genomic DNA from the 210 ovarian-cancer samples in the mutation-validation cohort. All unique somatic mutations detected in samples of ovarian clear-cell carcinoma, endometrioid carcinoma, and high-grade serous carcinoma are shown. Numbers 1 through 6858 below the schematic indicate the nucleotide (nt) position, starting with the A in the ATG start codon for ARID1A in position 1 (based on the sequence given in record number NM_006015.4 in Entrez Gene; also see Table 1 in the Supplementary Appendix). UTR denotes untranslated region.

Figure 2. Results of Immunohistochemical Analyses of BAF250a Expression

The percentages of tumors (with number and total number in parentheses) from three subtypes of ovarian cancer — clear-cell carcinoma (CCC), endometrioid carcinoma (EC), and high-grade serous (HGS) carcinoma — from the discovery and mutation-validation cohorts that showed loss of BAF250a expression are shown in Panel A for samples with and samples without ARID1A mutations and in Panel B for samples in the discovery and mutation-validation cohorts and samples in the immunohistochemical validation cohort. The rate of BAF250a loss was higher among CCC specimens with an ARID1A mutation than among those without an ARID1A mutation (P<0.001); the same was true for EC specimens (P = 0.02). The loss of expression was also consistently more common in CCC and EC (the two endometriosis-associated carcinomas) than in HGS carcinoma when assessed in the discovery and mutation-validation cohorts and again in the immunohistochemical validation cohort (Panel B), with P<0.001 for all comparisons. All P values were calculated with the use of Fisher’s exact test.

Figure 3. Analysis of Ovarian Clear-Cell arcinoma and Associated Endometriosis in a Study Patient

Panel A shows a section (hematoxylin and eosin [H&E]) on which a clear-cell carcinoma (black arrow) has arisen in an endometriotic cyst (white arrow). The same section, viewed at a higher magnification, shows regions of the clear-cell carcinoma and contiguous atypical endometriosis. A region of distant endometriosis from the same patient is also shown. Panel B shows the results of immunohistochemical staining of the epithelial portions of tissue specimens shown in Panel A for expression of BAF250a, hepatocyte nuclear factor 1β (HNF-1β), and estrogen receptor (ER). BAF250a immunoreactivity is lost in both the clear-cell carcinoma and the contiguous atypical endometriosis but is maintained in the distant endometriosis. Both regions of endometriosis differ from the carcinoma in their lack of HNF-1β expression (with weak expression in the contiguous atypical endometriosis) and maintenance of estrogen-receptor expression. Panel C shows sequencing chromatograms for the clear-cell carcinoma and polymerase-chain-reaction (PCR) clones of microdissected material from the contiguous atypical endometriosis and distant endometriosis, from which DNA was extracted. The carcinoma and contiguous atypical endometriosis show nucleotide variation corresponding to G6139T (as indicated with the dashed box); the tumor shows a heterozygous peak at that location, whereas the atypical endometriosis is homozygous for the substitution (in 17 of 42 clones). In contrast, the distant endometriosis shows wild-type sequence (in all 52 clones analyzed). None of the PCR clones from the distant endometriosis showed variation from the wild-type sequence.